doi 10. 15171/ijpni.2016.

06
International Journal of Phytocosmetics
and Natural Ingredients 2016;3:06 Original Article

Antimicrobial, Antioxidant and Anti-Inflammatory Assessment

of a Phytocosmetic Produced with Glycinin Peptides
Fernanda Corra da Silva Vasconcellos1, Adenise Lorenci Woiciechowski1, Carlos Ricardo Soccol1
1
Department of Bioprocess Engineering and Biotechnology, Bioprocess Laboratory, Federal University of Paran,
UFPR, Curitiba, PR, Brazil.

Correspondence to
Fernanda Corra da Silva
Vasconcellos
Email:
fernandacsv@gmail.com
Received 2 September 2016
Accepted 7 October 2016
ePublished 31 December 2016
Abstract
Phytocosmetics are increasingly gaining attention from consumers who search for alternatives
for the maintenance and protection of the skin. This article reports formulation of a phytocos-
metic derived from defatted soybean flour, an emulsion base that is hydrolyzed from the glycinin
protein (1000 g/g). Test for stability, microbiological control and biological activities antimicro-
bial (agar diffusion method), antioxidant (method of ABTS/TEAC radicals) and anti-inflamma-
tory (method of hyaluronidase enzyme) properties were conducted. The results indicated that
the phytocosmetic was stable and had low indices of microorganisms, according to Resolution
481/99. The bioactivity of the glycinin peptides was not altered or harmed by the other compo-
nents of the emulsion. For the antimicrobial activity, the bacteria E. coli, S. aureus and P. acnes
had values of 30.5 mm, 28 mm and 25 mm halos, respectively. For the antioxidant activity, the
result was of 25.1 TEAC and the anti-inflammatory activity was measured at 83.4% inhibition of
hyaluronidase enzyme. This study showed that the formulated phytocosmetic has great potential
for topical use; it is comparable to other anti-aging cosmetics for daily skin care with antioxidant,
anti-inflammatory and antimicrobial properties.
Keywords: Glycinin, biological activity, antimicrobial, antioxidant, anti-inflammatory, phyto-
cosmetic.

Introduction ties. 5,6,7,8

The main nutritional components of the soybean are the
proteins obtained from its by-product, defatted soybean
flour, which is derived from the soybean oil. The main
proteins present in the flour are the glycinin and -cong-
lycinin, corresponding to about 80% of the total proteins
in soybeans.1
Over the last decades, new molecules have emerged with
functions that are specific for skin care.2 Vegetable proteins
like wheat, corn, oatmeal, rice, sunflower and soy have be-
come important sources of peptides in the cosmetic field
and can be incorporated into lotions, gels and creams.3,4
Several peptides in plants are being identified and charac-
terized and are promising candidates for different applica-
tions for human health. Studies have reported that main
biological activities of these peptides are antioxidant, an-
timicrobial, anti-inflammatory, anti-tumor, anticholester-
olemic and antihypertensive. Glycinin consists of an acid
and basic subunits (molecular mass of 34 and 20 KDa re-
spectively), linked together by a disulfide bond, the basic
subunit is both hydrophobic and cationic and may be able
to react with bacterial cell wall and with acid subunit are
responsible for antioxidant and anti-inflammatory activi-
Nowadays, consumers are on a constant quest for a
younger, prettier, healthier appearances and are seeking
alternatives for skin care. This has caught the attention of
the cosmetic industry. Brazil is the third largest producer
of cosmetics in the world, behind the United States and
China. In 2014 the revenue from the cosmetic market
amounted to R$160 billion. 9
Phytocosmetics are cosmetics with vegetable active prin-
ciples that are incorporated into emulsion and are respon-
sible for the biological activity.10 The cosmetic emulsion
can have antimicrobial, antioxidant or anti-inflammatory
activities, and used on the face as an adjuvant for the con-
trol of acne and premature aging.
Acne is inflammation in the skin, which occurs due to an
imbalance in the sexual hormones, it is very common in
adolescence, but it can also occur during pregnancy and
menopause. The bacteria that normally inhabit the fol-
licles feed on the excess oil, forming colonies that cause
swelling, redness and, later, the eruption of the follicles.11
On the other hand, aging is a set of irreversible and inevi-
table physiological alterations, and it is a dynamic process
observed on the epidermis resulting from the modifica-

Please cite this paper as: Vasconcellos F, Woiciechowski A, Soccol CR. Antimicrobial, antioxidant and anti-inflammatory
assessment of a phytocosmetic produced with glycinin peptides. International Journal of Phytocosmetics and Natural Ingre-
dients. 2016;3:06. doi:10.15171/ijpni.2016.06.
Vasconcellos et al.
http://ijisonline.org/IJPNI Antimicrobial, Antioxidant and Anti-Inflammatory Assessment

tions that occur in the conjunctive tissue of the dermis. Accelerated Stability Testing (AST)
Skin aging is part of the changes that occur in different After the centrifugation, the AST was performed. This test
sectors of the organism, and it is divided into two types. consists of applying different temperatures with the goal
The intrinsic one, which results from the natural aging of of accelerating possible reactions among the components,
the body with the passage of years and is expected, pre- which must be observed. 15
dictable, inevitable, progressive, with no interference from Twenty grams of the phytocosmetic and the control
external agents and equivalent to the aging of all the or- (emulsion base without glycinin peptides) were put in
gans. In addition, the extrinsic type is due to smoking and transparent flasks with wide mouths and screw caps. The
physical, nutritional and mechanical factors.12 formulations were subjected to storage in the following
Low molecular weight peptides have been used in cos- temperature situations:
metics because they have different functions: cicatrizing, room temperature (indirect lighting): 25C 2C;
depigmentizing, regenerating the extracellular matrix, di- low temperature (cooler): 5C 2C;
minishing wrinkles, improving firmness and elasticity.13 freezer: -10C 2C;
Thus, incorporating glycinin peptides an emulsion and high temperature (heater): 45C 2C;
assessing the biological potential of this phytocosmetic is cycles: alternating temperatures -10C 2C and 45C
highly favorable for innovation in the field of cosmetics, 2C (24 h for each temperature).
mainly the consumer association against the use of ani- All samples analyses at 0; 5; 10 and 15 days. The parame-
mal-derived ingredients, which allowed vegetable pep- ters were analyzed involved possible alterations in aspect,
tides derivatives to become more important. 12 color, smell, pH value and separation of phases.16
Regarding the parameter aspect, the product must remain
Materials and Methods intact during all the days of analysis, maintaining the ini-
Obtaining the Glycinin Peptides tial appearance, except in high temperatures, freezer or
The peptides were obtained according to Vasconcellos et freezing and thawing cycles, when small alterations are ac-
al. 14 ceptable. Regarding the parameters color and smell, they
must be stable during the 15 days of testing and small al-
Developing the Emulsion terations are acceptable only in high temperatures.15
The emulsion base was produced according to Table 1. The separation of phases was observed visually, and for
The aqueous phase was heated to 75C and the oil phase to analyzing the pH values, 1g is taken and homogenized in
80C. The aqueous phase was transferred into the oil phase 9mL of distilled water, determining the pH value at room
with agitation until cooling. temperature.
Stock solutions of glycinin peptides were into the emul-
Table 2. Criteria for the assessment of the organoleptic parameters
sion in order to obtain a final concentration of 1000 g/g
of the cosmetic emulsion.
of sample.
Symbology Aspect, Color and Smell
Table 1. Components of the emulsion for producing the phytocos-
N normal, no alteration
metic.
S slightly different
Components Action Amount (%)
M different
Deionized water Vehicle q.s.p. (100)
I intensely different
Disodium EDTA Chelating 0.10
Propylene Moistening 2.00 Table 2 shows the criteria for the assessment of the phyto-
Methylparaben Preservative 0.50 cosmetic emulsion.
Cyclomethicone Emollient 1.00
Microbiological Control of the Phytocosmetic
Glyceryl Stearate Emulsifier 1.50
Performing biological control on non-sterile products
Carbomer Thickening 1.00 aims determination at microorganisms present in the
Cetyl alcohol Emulsifier 0.20 sample. For counting these microorganisms, 1g of the
Glycerin Moisturizing 2.50 phytocosmetic was dissolved and homogenized in 9mL of
sterile 0.1% peptone water. One mL of this solution was
Stability Study placed on petri dishes containing approximately 20mL
The formulation was assessed as recommended by the of agar nutrient for bacteria and 20mL of sabouraud agar
Cosmetic Products Stability Guide from the National for fungi. The dishes were incubated at 37C for 24 h for
Health Surveillance Agency.15 the analysis of the bacteria and at 25C for 7 days for the
analysis of fungi. After this period, the number of colo-
Centrifugation Test ny-forming units (CFU/g sample) was determined.15
Five grams of the phytocosmetic were centrifuged three
times at 3,000 rpm for 30 min with temperature of 25C. Biological Activity
After this period, the formulation was assessed as to the For the antimicrobial activity, the agar diffusion test was
separation of phases, indicating stability or not. performed on the strains Escherichia coli ATCC 26922,

2 International Journal of Phytocosmetics and Natural Ingredients 2016, 3:06

Vasconcellos et al.
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Staphylococcus aureus ATCC 25923 and Propionibacte-
rium acnes ATCC 6919, which inhabit the skin and can
cause skin infections as acne.17,18,19
The experimental design involve phytocosmetic with gly-
cinin peptides (1000 g/g), antibiotic tetracycline (30 g/
mL) as a positive control and sterile distilled water and
emulsion base without glycinin were negative controls.
Briefly, the peptide suspension were sterilized with 0,22
m membrane (Millipore, Billerica, MA, USA) and bac-
terial suspension were adjusted by 0,5 MacFarland com-
parasion. Petri dishes containing culture medium and
bacterial suspensions were prepared and after agar solid-
ified, wells were made and 150 L each sample, positive
and negative control were added. Incubated at 37 C for a
time appropriate each strain and the antibiosis effects were
determinate by measuring inhibited halos (mm).
The hyaluronidase enzyme method was used for the an-
ti-inflammatory activity.20,21,22 The phytocosmetic with
glycinin peptides (1000 g/g) was homogeneized with 500
/L hyaluronic acid solution (1,2 mg hyaluronic acid/mL
0,1 M in acetate buffer, pH 3,6 and 0,15 M in NaCl) was
incubated at 37C with 50L hyaluronidase enzyme for
40 min. After 0,8 M potassium tetraborate was added and
incubated at boiling for 3 min, 0,3 mL p-dimethylamino-
benzaldehyde was homogeneized for 20 min at 37C. The
positive control was dimethylsulfoxide, a topic anti in-
flammatory and negative control were sterile distilled wa-
ter and emulsion base without glycinin. The results were
mesuared in 585nm absorbance and the anti-inflammatoy
activity was mensured percentage of enzyme inhibition.
The antioxidant activity was used the ABTS free radical
method.23,24
ABTS solution (200 L), Trolox (50 L) and suspensions
of phytocosmetic with glycinin peptides (1000 g/mL)
and emulsion base without glycinin were added to 96 well-
plate and incubated in the dark for 6 minutes at room tem-
perature, after the absorbance at 734 nm was mensured
with microplate reader (Power Wave XS, Biotek, Winoos-
ki, VT, USA). The antioxidant activity expressed as Trolox
equivalent antioxidant capacity (TEAC).
Statistical Analysis
The results obtained in this study were expressed by the
mean standard deviation of triplicates. Differences were
analyzed using ANOVA followed by the Tukeys test and
the Student-t test for comparison between means when
necessary. Differences were considered significant with p
value < 0.05.
Results and Discussion
Centrifugation Test
This procedure is efficient for determining the instability
of emulsified products, since the simulation of an increase
in the force of gravity can separate the components that
have different densities.25
In this study, there was no separation of phases in the con-
trol group and the phytocosmetic group, suggesting the
emulsion is stable when facing gravitational stress.
International Journal of Phytocosmetics and Natural Ingredients 2016, 3:06
Antimicrobial, Antioxidant and Anti-Inflammatory Assessment
The absence of separation of phases demonstrates that in
normal conditions of gravity and at room temperature, the
phytocosmetic is physically stable; this occurred in this
study.26
Accelerated Stability Test
As to the organoleptic parameters aspect, color, smell and
separation of phases, there were no relevant alterations
in all stress conditions during the 15 days of analysis.
The variables high temperature and cycles caused subtle
changes only in the aspect of the phytocosmetic and the
control, as can be seen on Table 3. This change may have
occurred due to the evaporation of water when the phyto-
cosmetic and the control were in the heater.16,10
Table 3. Assessment of the organoleptic characteristics (color, smell
and aspect) and separation of the phases of the phytocosmetic and
control.
Variables/ Days 0 5 10 15
Room temperature N N N N
Low temperature N N N N
Freezer N N N N
High temperature N N N S
Cycles N N N S
For the parameter pH stability was observed from the 10th
day of analysis, as shown on Figure 1. The control behaved
similarly to the phytocosmetic, which demonstrates that
adding glycinin did not alter the properties of the com-
ponents of the emulsion. The formulation presented val-
ues between 5.8-5.9 compatible with the skin pH which
is around 5.0-6.0.27 All the conditions tested during the
15 days did not present significant differences (p< 0.05),
which confirms the stability of the phytocosmetic pro-
posed in this study.
Figure 1. pH variation in the phytocosmetic and control during the 15
days of analysis in the accelerated stability test.
Microbiological Control of the Phytocosmetic
The Resolution RDC 48128 establishes the limits for
the microbiological control in personal hygiene products,
perfumes and cosmetics. The phytocosmetic produced in
this study, according to this resolution, is on level II, that
is, products that have specific indications and have charac-
teristics that demand safety and/or efficiency proof, as well
as information, warnings, instructions for use and restric-
tions. Thus, the maximum allowed contamination by aer-
3
Vasconcellos et al.
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Table 4. Antimicrobial activity of the phytocosmetic and tetracycline
antibiotic*
Strains Halos (mm) Phytocosmetic Tetracycline
E.coli 30.50.50A 32.50.50Aa
S. aureus 28.00.20Bb 30.00.10Bb
P. acnes 25.00.30Cc 27.50.20Cc
*Mean standard deviation of the triplicates
Uppercase letters show significant differences between phytocos-
metic and antibiotic.
Lowercase letters show significant differences between the strains.
Tukeys range test (p <0.05)
obic microorganisms is of 5 x 103 CFU/g or mL of sample.
The total number of microorganisms in the phytocosmetic
and the control was absent for bacteria and with indices
lower than 4 CFU/g for fungi. These results are inside the
limit recommended by the resolution, and there was no
need to isolate the colonies and identify them.
Biological Activity
Antimicrobial Activity
The results of the agar diffusion test for the bacteria S.
aureus, E. coli and P. acnes can be seen on Table 4. Gly-
cinin peptides showed antibiotic activity, all strains were
susceptible. The positive control showed halos with media
30,0 mm and negative control did not effect without halos.
The concentration 1000 g/g glycinin peptides was similar
effect that tetracycline antibiotic (30 g/mL). Futhermore,
the high concentrations of glycinin peptides are required
to produce like effect to that commercial antibiotic.
Peptides kill bacteria by permeabilization through the
formation of stable pores, as well as by membrane thin-
ning or micellization in a detergent-like manner and pos-
itive charges peptides facilitate interaction with negative
charges bacteria phospholipid containing.29,30
The gram- negative bacteria are susceptible peptides,
because the lipopolysaccharides that have high nega-
tive charge and facilitate linkage to cationic peptides and
gram- positive bacteria have negative charge in teichoic
and lipoteichoic acids in peptidoglycans, therefore, both
bacterial were susceptible to peptides.31,32
Anti-inflammatory Activity
The phytocosmetic has 83.4% of anti-inflammatory activ-
ity. The peptides action in the anti-inflammatory process
inhibiting hyaluronidase enzyme degrades hyaluronic
acid, present in the skin, in fragments in which high po-
tential process inflammation, increasing permeability and
decreasing the firmness and elasticity of skin. 33,34
Antioxidant Activity
The antioxidant activity found in the phytocosmetic with
the ABTS radical method was of 25.11.75 ABTS. The
standard curve of the trolox is on Figure 2. Peptides orig-
inating from milk and soybeans have been shown to ex-
hibit antioxidant activity due abilities to inactivate reac-
tive oxygen species (ROS), scavenge free radicals, chelate
4 International Journal of Phytocosmetics and Natural Ingredients 2016, 3:06
Antimicrobial, Antioxidant and Anti-Inflammatory Assessment
Figure 2. Standard curve of the trolox against the ABTS radical.
pro-oxidative transition metals, reduce hydroperoxides.35
These peptides act on the stratum corneum of the skin (su-
perficial layer), improving hydration, elasticity, expression
lines, improving the antioxidant ability, regenerating fibro-
blasts and reducing acne inflammation. There is evidence
that ROS are involved in the process of skin aging, and the
topical application of cosmetics with antioxidant proper-
ties has certified biological effects on the ROS. 36,37,38,4
Alanine, aspartic acid, phenylalanine, histidine, tyrosine,
methionine, cysteine are responsible antioxidant capaci-
ty peptides.39,40 And the glycinin constituted in aspartic
acid, threonine, serine, glutamic acid, proline, glycine,
alanine, cysteine, valine, methionine, isoleucine, leucine,
tyrosine, phenylalanine, histidine, lysine and arginine
amino acids.14
However, the amino acids arginine, lysine, histidine (cat-
ionic), alanine, leucine, isoleucine and methionine (hy-
drophobic) are responsible for the connections with the
epithelial cells of the stratum corneum, which have nega-
tive charges, increasing their permeability and easing the
passage of the phytocosmetic.41,42,43
The phytocosmetics play an important part in the cosmet-
ic field due to their relevance and high efficacy.44 The use
of bioactive peptides in this field is promising, due to the
great versatility and diversity of biological activities.45
As was observed in study that demonstrated the capacity
of the use of defatted soybean flour as a source of proteins
for the formulation of a phytocosmetic with great bioac-
tive power.
Conclusion
The stability study showed that the phytocosmetic was
stable when it came to environmental changes during the
period of analysis and it did not present relevant indices of
external contamination by microorganisms.
The phytocosmetic was efficient regarding the antimicro-
bial, antioxidant and anti-inflammatory biological activ-
ities. It demonstrated the great potential of peptides de-
rived from glycinin as a source of vegetal raw material to
be used in the cosmeceutical field.
However, this phytocosmetic requires more research and
tests before its topical use.
Vasconcellos et al.
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Competing Interests
The authors declare no competing interests.
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